CN109456985B - Pathogenicity-related botrytis cinerea gene BcMBF1 and application thereof - Google Patents

Abstract

The invention provides a botrytis cinerea gene BcMBF1 related to pathogenicity and application thereof, belonging to the technical field of microbial genetic engineering, wherein the DNA sequence of the gene BcMBF1 for controlling hyphal branching and pathogenicity of botrytis cinerea is shown as SEQ ID No. 1 and consists of 1398 nucleotides; the amino acid sequence of the protein coded by the BcMBF1 gene is shown in SEQ ID No. 2 and consists of 465 amino acids; the BcMBF1 gene can be applied to the field of plant gray mold resistant genetic engineering; the pathogenicity of the botrytis cinerea is deficient by deleting, mutating or modifying a protein BcMbf1 for controlling hypha branching and pathogenicity of the botrytis cinerea, and the botrytis cinerea can be used as targets for designing and screening anti-botrytis cinerea medicaments.

Description

Pathogenicity-related botrytis cinerea gene BcMBF1 and application thereof

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to discovery of a novel gene for controlling pathogenicity of fungi in the field of plant protection and application of a protein coded by the novel gene.

Background

Botrytis cinerea (A), (B), (C), (B), (C)Botrytis cinerea) Also commonly known as botrytis cinerea, a fungus belonging to the phylum Ascomycota (Ascomycota), is the causative bacterium of gray mold and infects over 1400 plants, including almost all vegetables and fruit trees. Hosts can be attacked from the seedling stage, the fruiting stage to the storage stage, all parts of plants can be infected by botrytis cinerea, typical symptoms of leaf attack are shown as V-shaped disease spots, flowers are mainly shown as rotten and withered, and fruits are mainly shown as rotten and fallen. The occurrence and spread of the disease have close relation with the humidity and temperature of the environment, and the disease is serious when the relative humidity is more than 90 percent at the temperature of between 20 and 23 ℃. Therefore, the gray mold is of a low-temperature and high-humidity typeDiseases, which are most likely to occur in rainy seasons or in protected field production, cause economic losses of up to $ 100- $ 1000 billion annually in the world. Due to the wide host range and serious production hazard and the mature related molecular research technology, the botrytis cinerea has become one of the most important model plant pathogenic fungi and is widely researched.

Botrytis cinerea is a typical dead body nutritional pathogenic fungus, can generate a plurality of pathogenic factors to participate in pathogenesis, mainly comprises cell wall degrading enzymes, cutinase, toxin, plant hormone, enzymes resisting host reaction, small RNA (ribonucleic acid), small molecular substances and the like, and the factors cooperate with each other to ensure that the Botrytis cinerea can kill host cells and decompose dead host tissues as nutrition. Under natural conditions, botrytis cinerea mostly takes conidia as a primary infection and secondary infection source of an infected host. Botrytis cinerea often attaches to plant disease residues as mycelia, conidia or sclerotia, or overwintering and overwintering in soil, and becomes the primary infection source of the next growing season. When conditions are appropriate, sclerotia germinate to produce mycelia and conidiophores, and produce a large number of conidia. Mature conidia can be spread by wind, rain, irrigation and drainage water, farming operation and the like. Under the condition of low temperature and high humidity, conidia germinate to form a germ tube, the end of the germ tube slightly expands to develop into an attachment cell or further form an infection structure such as an infection pad and the like, and the infection structure mainly invades from decayed floral organs, wounds and necrotic tissues.

When the conidia of the botrytis cinerea with high concentration infect hosts, the disease is rapidly developed, and the conidia mainly invade through attachment cells formed at the top ends of the germ tubes; the rate of invasion through the top of the germ tube is reduced with a decrease in spore concentration, and the rate of attack is correspondingly delayed by 1-4 days, when invasion is primarily through attached cells or invaded pads developed by hyphae. After the botrytis cinerea invades host cells, the host cells are directly challenged by hostile environment in host tissues, pathogenic bacteria must be quickly adjusted to inhibit defense reaction of plants on one hand and actively adapt to physical and chemical environments in the host cells on the other hand, and thus the botrytis cinerea is expected to successfully parasitize the plants on both sides. Botrytis cinerea achieves the above-mentioned goals to a large extent by changing its own metabolic pathways and secreting relevant effector factors (e.g., toxins), but the molecular mechanisms of genes, proteins, and metabolites and their regulation involved in the corresponding processes are still poorly understood. The field is deeply researched, the key factor of botrytis cinerea for adapting to the internal environment of a host is identified, the molecular mechanism of botrytis cinerea, which is a dead body nutritional pathogenic fungus, is disclosed, and protein serving as a fungicide action target can be found out possibly, so that the theoretical and technical basis is laid for developing efficient medicaments for preventing and treating botrytis cinerea and other similar diseases.

MBF1Is a gene with unknown function peculiar to botrytis cinerea, and homologous genes of the botrytis cinerea are not found in other species. By analysing Botrytis cinereaMBF1The pathogenic function of the gene, and the evaluation of the effect of the gene in the development and pathogenic process of botrytis cinerea are beneficial to identifying potential prevention targets, and the gene is used for screening novel botrytis cinerea killing medicaments.

Disclosure of Invention

The purpose of the present invention is to provide a gene for controlling hyphal branching and pathogenicity and a protein encoded by the gene.

The gene for controlling hypha branching and pathogenicity is derived from botrytis cinerea and is named asBcMBF1The DNA sequence is shown in SEQ ID No. 1. The DNA sequence isBcMBF1An open reading frame of a gene consisting of 1398 nucleotides, wherein there is no intron sequence.

The invention providesBcMBF1The amino acid sequence of the protein coded by the gene is shown as SEQ ID No. 2, and the sequence consists of 465 amino acids.

Genes from Botrytis cinerea for controlling hyphal branching and pathogenicityBcMBF1Can be applied to the field of plant gray mold resistant genetic engineering.

Genes controlling hypha branching and pathogenicity from Botrytis cinereaBcMBF1The encoded protein is subjected to deletion, mutation or modification, so that the pathogenicity of the encoded protein is deficient, and the encoded protein can be used as a target to be applied to designing and screening of anti-botrytis cinerea medicaments.

The invention provesBcMBF1The deletion or mutation of a gene or genes,results in a significant reduction in the pathogenicity of Botrytis cinerea, which indicatesBcMBF1The gene is necessary for gray mold of crops caused by gray mold pathogen. Therefore, screening compounds capable of preventing the gene expression and the protein expression, modification and positioning of the gene can effectively control the occurrence of gray mold, thereby being beneficial to developing novel bactericides, namely the bactericide provided by the inventionBcMBF1 is that: the expression of the gene and the expression, modification and positioning of the protein product coded by the gene can be used as an important candidate target site for the design and screening of the botrytis cinerea resistant medicament.

Drawings

FIG. 1 is a drawing ofBcMBF1Schematic domain prediction of proteins

Wherein: no conserved functional domains were found; transmembrane regions (indicated by arrows in the figure), predicted to be transmembrane proteins;

FIG. 2 shows Botrytis cinereaBcMBF1Schematic representation of knockout strategy of genes (Gene replacement by homologous recombination)

Wherein: b05.10 is a Botrytis cinerea wild type strain, and pMFB1-KO is a knock-out carrierBcmbf1Is composed ofBcMBF1Deletion mutants of genes; primers P1 and P2, P5 and P6 are used for amplification respectivelyBcMBF1Upstream and downstream sequences of the gene, which serve as homology arms for the knockout vector; primers P3, P4, P7 and P8 are used for verifying the mutant and the complementary strain;

FIG. 3 is a drawing showingBcMBF1PCR (polymerase chain reaction) verification electrophoretogram of gene deletion mutant and genetic complementation strain

Wherein: p7, P8, P3 and P4 are used as primers, and the corresponding binding positions are shown in figure 2; m1 and M2 areBcMBF1A gene deletion mutant; ΔBcmbf1the-C is transferred into the whole on the basis of the mutant M1BcMBF1A complementary strain of the gene;

FIG. 4 is a drawing showingBcMBF1Deletion mutant of gene, wild strain B05.10 and complementary strainBcmbf1-CComparative photograph of culture characteristics

Wherein: the culture medium is PDA, and is cultured at 20 ℃, and the pictures are observed and taken 3 days after inoculation; WT is Botrytis cinerea wild type strain B05.10, and the numbering of the other strains is as described above.

FIG. 5 is a drawing showingBcMBF1Deletion mutant of gene, wild strain B05.10 and complementary strainBcmbf1-CMicroscopic contrast picture of spore growth

Wherein: the spores are produced by inoculating the corresponding strains on a PDA culture medium for 10-15d, inoculating 1/2PDB culture medium on a glass slide, and evaluating after inoculating for 8h, 10h and 14 h.

FIG. 6 is a drawing showingBcMBF1Quantitative analysis of hypha branching between gene deletion mutant and control Strain

Wherein: the inoculation method is the same as above, measurement statistics is carried out after 8h, 10h and 14h of inoculation, and the measurement statistics is converted into relative sizes. Indicates significant differences at p <0.001 levels.

FIG. 7 is a drawing showingBcMBF1Comparative pictures of pathogenicity of gene deletion mutant, wild-type strain and complementary strain

Wherein: the selected host is kidney bean, and the spore is inoculated with the excised leaf. Evaluation is carried out 48h and 72h after inoculation;

FIG. 8 is a drawing showingBcMBF1Quantitative analysis schematic diagram of lesion size generated by infecting host with mutant and control strain of gene

Wherein: the inoculation method is the same as above, the areas of the leaf lesions are measured and calculated 48h and 72h after inoculation, and the areas are converted into relative sizes. Indicates significant differences at p <0.001 levels.

Detailed Description

In order to better describe the invention, the following is further illustrated by specific examples, the methods of which, unless otherwise specified, are conventional.

Example 1BcMBF1Correlation analysis of genes

GeneBcMBF1Is a gene identified by the research and development team through analyzing T-DNA marked botrytis cinerea pathogenic defect mutant. Botrytis cinerea (Fr.) KuntzeBcMBF1The open reading frame of the gene consists of 1398 nucleotides and has no intron. The coded protein product consists of 465 amino acids, and domain analysis shows that the BcMbf1 protein does not detect a conserved functional domain and is a protein with unknown function; the sequence contains a transmembrane regionWas detected as a transmembrane protein (see FIG. 1).

Example 2BcMBF1Gene knock-out

1) Construction of knockout vectors

Primers P1 (5'-ACTAGTCTGTGCCTCGTGGTGAAGAT-3') and

p2 (5'-GGTACCTGATGGCAATCCGTTGAAGT-3'), amplified using genomic DNA of Botrytis cinerea strain B05.10 as templateBcMBF1The upstream 604bp fragment of the gene is adopted

P5 (5'-GTCGACATGGGTGGTTTATTCTATTTGC-3') and

p6 (5'-CTGCAGGAGTGGATGGCTCGTGAAG-3') amplification of Botrytis cinereaBcMBF1The 655 bp fragment at the downstream of the gene, the reaction system is: 10 mmol/L dNTP mix, 0.5 μ L; 10 × PCR buffer, 2.5 μ L; 1 muL (10 mumol/mL) of each upstream and downstream primer; template DNA, 1 μ L; Ex-Taq, 0.2 μ L (5U); ddH₂O, 18.8 muL; the amplification procedure was: pre-denaturation at 94 ℃ for 3 min, followed by (1) denaturation at 94 ℃ for 50 sec; (2) annealing at 58 ℃ for 50 seconds; (3) extension at 72 ℃ for 60 seconds; (4) circulating for 30 times; (5) extension at 72 ℃ for 10 min. Cloning the two DNA amplification products to pXEH vectorSpeI、KpnI site andSal I、Psti site, constructed as knock-out vector pMBF1-KO (see FIG. 2), and sequence verified.

Botrytis cinerea (A) and (B) used in the inventionBotrytis cinerea) Strain B05.10, purchased from the American Fungal genetic Material Collection (FGSC), from which other personnel may purchase the strain if desired, the relevant deposit information being as follows:

the strain number is as follows: FGSC 10317.

The address of the collection center: fungal Genetics Stock Center, Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center, Manhattan, KS 66506 USA.

Website address: http:// www.fgsc.net/scripts/StrainSearchReturnPage. aspOrgID = 23812.

2) Transformation of Botrytis cinerea

a. Cultivation of Agrobacterium

A single colony of Agrobacterium tumefaciens strain Agl-1 containing a binary vector pMBF1-KO is picked and inoculated into an MM liquid medium (dipotassium phosphate 0.205%, potassium dihydrogen phosphate 0.145%, sodium chloride 0.015%, magnesium sulfate heptahydrate 0.05%, calcium chloride hexahydrate 0.01%, ferrous sulfate heptahydrate 0.00025%, ammonium sulfate 0.05%, glucose 0.2%) containing 50. mu.g/ml kanamycin and 10. mu.g/ml rifampicin, and subjected to shaking culture at 250 rpm and 28 ℃ for 48 hours; centrifuging at 4000 rpm for 5 minutes, discarding the supernatant, resuspending an IM liquid culture medium (dipotassium hydrogen phosphate 0.205%, potassium dihydrogen phosphate 0.145%, sodium chloride 0.015%, magnesium sulfate heptahydrate 0.05%, calcium chloride hexahydrate 0.01%, ferrous sulfate heptahydrate 0.00025%, ammonium sulfate 0.05%, glucose 0.2%, 200 μ M AS, MES 0.854%, and glycerol 0.5%), centrifuging at 4000 rpm for 5 minutes, and discarding the supernatant; the IM medium was resuspended, cultured at 28 ℃ and 250 rpm for 6 hours with shaking, and pre-induced.

b. Spore-forming culture of botrytis cinerea

Selecting B05.10 strain, coating a small amount of spore on PDA culture medium (potato 20% boiled and filtered, glucose 2%, agar 1.5%), culturing at 28 deg.C for 8 hr to make spore rapidly germinate, transferring to 20 deg.C for culturing for 3-5 days, after thallus surface is covered by gray spore, scraping and collecting spore with IM liquid culture medium, observing with microscope, regulating spore concentration to 1 × 10 with hemocytometer⁶/mL。

c. Co-culture of agrobacterium tumefaciens and botrytis cinerea conidia and screening of transformants

Mixing an agrobacterium liquid and a botrytis cinerea spore liquid which are induced in an IM liquid culture medium for 6 hours in advance in the same volume, adding AS to enable the final concentration to reach 500 mu M, uniformly mixing, then uniformly coating the mixture on an IM culture medium paved with cellophane according to 250-350 mu L/dish, and culturing for 48 hours in the dark at 22 ℃; after the co-cultivation was completed, the cellophane was transferred to PDA medium containing 100. mu.g/mL hygromycin, and the cultivation was continued under the same conditions. After 4-7 days, the expanded colonies are picked up and placed on a screening medium containing the same antibiotics.

3) Validation of deletion mutants

Two pairs of primers were selected for screening of the transformants by PCR amplification. The amplification results are in accordance withIs determined asBcMBF1Gene deletion mutants: primers on the genome outside the upstream homology arm

Primers for P7 (5'-AGGGACATCGGTTTCAGCAC-3') and hygromycin resistance gene

The P8 (5'-ACAGACGTCGCGGTGAGTTCA-3') pair can be amplified to a recombinant fragment of the expected size (1.3 kb); and coding region primer P3 (5'-CGCAACTCTGATGATGTGGA-3') and

no amplified band was observed for P4 (5'-GAGCGTGTAATAAATGGAAGCA-3') (0.8 kb fragment was amplified in the wild type strain). As a result, 2 strains were selected from the transformantsBcMBF1Gene deletion mutants: m1, M2 were used for subsequent functional analysis (see FIG. 3).

Example 3BcMBF1Genetic complementation of Gene deletion mutants

Amplification of Botrytis cinerea with primers C-F (5'-AAAGATCAAAGGATCGAATTCAGAAAGAGGCGATTGTGAAGT-3') and C-R (5'-CCGGGTACCGAGCTCGAATTCATATGTTAGCGAACGAAGCAG-3')BcMBF1The gene with the total length of 3208 bp (comprising a promoter, an open reading frame and a terminator) is firstly cloned to a pMD18-t vector and then is subcloned to a pXEB vector (containing a glufosinate-ammonium resistance gene)EcoR I locus to construct a genetic complementary vector pMBF 1-ko-c. The vector was verified by sequencing to have no amino acid mutations. This complementary fragment was transferred into Agrobacterium-mediated transformation as described above using 200. mu.g/mL glufosinate for selectionBcMBF1Obtaining the genetic complementation strain Δ in the genome of the gene deletion mutant M1Bcmbf1-C. The primers P7 and P8, P3 and P4 used in the mutant verification were selected for PCR amplification, and the results are as expected (see FIG. 3): the complementary strain is the same as the mutants M1 and M2Bcmbf1-COriginal one of the originalBcMBF1Replacement of the Gene with the hygromycin resistance GeneHPH(P7 and P8 were positive), but additionally had a subsequently transferred geneBcMBF1Gene (the coding region primer P3 was positive similarly to the amplification result of P4).

Example 4BcMBF1Action of gene in hypha growth process of Botrytis cinerea

Evaluation by platingBcMBF1Hyphal growth of the mutantsAnd the like, and the related phenotype. Taking 10 uL of PDB spore suspension (1X 10) of the strain to be detected⁶ ml^-1) Inoculating in the center of PDA culture medium, and culturing at 20 deg.C in dark. After three days, the colony morphology of the mutant is basically normal, and the colony size has no obvious difference with the wild type and the complementary strain, which indicates thatBcMBF1Genes that are not essential for the extension of hyphae of Botrytis cinerea (see FIG. 4). Microscopic observation shows that hypha branches begin to increase after conidia of the wild strains germinate along with the extension of the culture time, and the branching capacity of the mutant is obviously reduced (see figure 5); after spore culture for 14h, the hypha branching ability of the mutant is only about 50% of that of the wild type (see FIG. 6). The branching ability of hyphae of the complementary strain can be restored to a wild type level (see fig. 5 and 6), and the results show that the gene plays an important role in the branching process of the hyphae of the botrytis cinerea. Therefore, we will name it asBcMBF1（ B . c inerea mycelial branching-related factor 1）。

Example 5BcMBF1Function of gene in pathogenicity of botrytis cinerea

Evaluation by in vitro leaf inoculationBcMBF1The pathogenic changes of the mutant. Collecting mature leaf from greenhouse cultured semen Phaseoli vulgaris plant, placing in container horizontally, and collecting 15 μ L of PDB spore suspension (1 × 10)⁵ ml^-1) And (3) dotting the strain on leaf surfaces, carrying out moisturizing dark culture at 20 ℃, and evaluating the pathogenicity of the strain to be detected in 48h and 72h respectively. The experimental results show that the high-temperature-resistant material,BcMBF1the mutant is obviously delayed in onset compared with the wild type, although the lesion is still formed, the lesion area is obviously smaller than that of the wild type and is difficult to expand, and the pathogenic capability of the complementary strain is basically restored to the wild type level (see figure 7). The area of the leaf scab is measured and calculated, and the wild type scab is found to be formed at 48h,BcMBF1the mutant did not develop disease, and the mutant developed disease at 72h, but the lesion area caused by the infection was only about 30% of that of the wild type (see FIG. 8). The results of this study show that,BcMBF1participates in the pathogenic process of Botrytis cinerea to plants, is necessary for Botrytis cinerea to infect hosts, ifThe gene or the protein coded by the gene loses activity, and the disease-causing capability of botrytis cinerea infecting hosts is seriously damaged.

Sequence listing

<110> Jilin university

<120> botrytis cinerea gene BcMBF1 related to pathogenicity and application thereof

<160>2

<211>1398

<212>DNA

<213> Artificial sequence

<400>1

1 ATGCCCACCA AAAACCTTCG AGAGATCGAG CCCAAACGCA AGGGACGAGC CCCAAGCGCA

61 AGTGCAAGTG AGTCTGATTC TAGTTCAGAT GACGGCAAAC AAACAGACCC TGTTGGCTCC

121 GGCGGCTCAT CGCTTGAAGG CTCTCACGCA ACTCTGATGA TGTGGATAGG ACTCCTTCTG

181 CTGATTTTGT TTCTGAATTG GGCATATATG ATACCCTATA TGACCCGATG GGTCCATCAT

241 GAGAAACTAC TCAGAAGTGG TTACAATGTC AAACTCATCA CCCCAAGTTG TGTATCTGCA

301 ATTCCGTTGT TATACAAGGA TATCACGTAC AACGCAACCA AAAAGGAAGG AGACATATTT

361 CGGGGACGAT TGGATACATT TCCAGGCAAC ACGCTTTTGA CTTTCAACAG GTACATTAAT

421 GAGATGCCAG ACTACCCGCA AGGCCGCCAA TTCCCGAAAC TATTTGAGGA TGTACAAGCT

481 GATGCTGACG AGATATATCA TGAATATATG GCATATTATG ATCTGGTTCG CCGACACACG

541 GAAGATGCAA TGCTACTCAC AAATGCCGTG ATAGAGGATT TGAACGAACT TATTTCTGAT

601 GTTGGACTCG TCAAAACTTC AAATTTCAAA AAACGGATAA TCAACCGCTT CTATTGGAGA

661 TATCTTTCCA AGAAGCCGAT AAGATCAAGA CTGATTAGAC AAATATTCAT TGAATATCTG

721 AAAGGGTTAC AAAAAGGATA TGTGCTACCG CAGTCAAATT CCCATCTGAA AAAATCCGTG

781 GGGTTACATG AAGTCATCGA GACAGGCAAT GAATTGTTGC CCAAGTTTGA AGAGGTCAAA

841 GATGCAAGTC GAGCGCTACG ATTCACTGTA GAGTTTGGCA TTTACAAAGT CTTGACAGAT

901 CAGAATTTGA CAAATCCTGC ATTTGTACCT CGCTCAGAGA TTTTAAGAAT GCTTCCATTT

961 ATTACACGCT CAAACAGCAG AGCTGGAAGA GGACATGACA ATGATACAAT CGGTTATATG

1021 ACCTATTGGC TCGATAATGA ATTTGCGGCC GGTGAAAATG TGCGTGAAGT GGAGATGCCC

1081 AACTCAGTCC GCCAATTTCA GCAAGCACTA GTCGACATTA CGAAACAGGT TTCAGAACTC

1141 GTTTATGATT TTTCGGATGA GAAGAATGGA ATTCAGGAGC TTTCATCGAC TGTATGTTAC

1201 ATTGATAAAT TAGAAAAAGC ACTCAGAGAT CTGGCCTTTC ATCATAACTT CGATTCAATA

1261 TTCCCAGGTC ACCCAAATCG TCCGAGAGCA ATTTGGAAGG TTCATAAATG TATTTGGGAA

1321 CGAGAAAATA GGGGATCCTT GAAAGAAACG ACTCATTGTG GCAAGCTTGA GGCGTTGATA

1381 AAGGAAAAGG TGTATTGA

<210>2

<211> 465

<212>PRT

<213> Artificial sequence

<400>2

1 MPTKNLREIE PKRKGRAPSA SASESDSSSD DGKQTDPVGS GGSSLEGSHA TLMMWIGLLL

61 LILFLNWAYM IPYMTRWVHH EKLLRSGYNV KLITPSCVSA IPLLYKDITY NATKKEGDIF

121 RGRLDTFPGN TLLTFNRYIN EMPDYPQGRQ FPKLFEDVQA DADEIYHEYM AYYDLVRRHT

181 EDAMLLTNAV IEDLNELISD VGLVKTSNFK KRIINRFYWR YLSKKPIRSR LIRQIFIEYL

241 KGLQKGYVLP QSNSHLKKSV GLHEVIETGN ELLPKFEEVK DASRALRFTV EFGIYKVLTD

301 QNLTNPAFVP RSEILRMLPF ITRSNSRAGR GHDNDTIGYM TYWLDNEFAA GENVREVEMP

361 NSVRQFQQAL VDITKQVSEL VYDFSDEKNG IQELSSTVCY IDKLEKALRD LAFHHNFDSI

421 FPGHPNRPRA IWKVHKCIWE RENRGSLKET THCGKLEALI KEKVY

Claims (1)

1.BcMBF1Gene in reducing botrytis cinerea (Botrytis cinerea) Use in pathogenicity, characterized in that it is in particular by knock-outBcMBF1Genes to reduce pathogenicity, saidBcMBF1The DNA sequence of the gene is shown in SEQ ID No. 1.

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